detection of phosphorylated proteins on WB - how stable is phosphorylation once transferred on WB membrane? (Feb/09/2006 )
Hi
i had Huh7 cells stimulated with Collagen and fibronectin and wanted to detect phosphorylated proteins using Upstate antibody against phosphotyrosines. but no band was detected! [I had no problem doing this with tissue lysate though...]: i find it hard to believe that there are no proteins that are phosphorylated in the cells! I loaded ~20ug of total protein to each well-enough protein, isn't it? 
could this be because i left the membrane (air dried and wrapped in gladwrap, stored overnight in fridge to do stainins the next day)? could the phosphorylations be very unstable that became hard to detect?
does anyone know or have any suggestions for this, please? 
No, phosphorylation is stable few days. I used to block overnight and still detect something the day after, You should still see something.
What did you use as phosphatases inhibitors in your lysis buffer?
What did you use as phosphatases inhibitors in your lysis buffer?
I used the phosphatase inhibitor cocktail from Sigma!? (and it worked really good when i was using it with the tissue homogenate).
-if you have add some Na3VO4 (orthovanadate) to inhibit tyrosine-phosphatase, maybe check again your Ab, i have the same problem with an anti-pTyr (4G10)-Po from BD which has completely lost ist activity in the fridge in 2 weeks!
-maybe 20µg of proteins are too much, i generally do not put more than 5µg for pTyr WB...
-as a positive control, incubate your cells in Na3VO4 200-500µM for O/N!
-did you used the same blocking solution than in the first experiment? I generally use T- or P-BS/Tween 0.5%/BSA 5%
-for storage of membrane (nitrocellulose in my case), always in TBS/Tween, never dried!
Sébastien_
agree with seb....if you r doing everythng else right i.e. protease inhibitors and etc...most likely it is the drying of the membrane. I store my pvdf in ttbs (seal it in a kolpakwith ttbs)
I agree with seb and pria,
actually I never dry my membrane to blot it later (only for autoradiography ).
What works however, is to freeze the membrane (in the substrate buffer, after X ray film exposure) at -80°C. I was lazy to do something else, and finally had to reuse one of these membrane. It worked.
But I never tried with a dried one.
actually I never dry my membrane to blot it later (only for autoradiography ).
What works however, is to freeze the membrane (in the substrate buffer, after X ray film exposure) at -80°C. I was lazy to do something else, and finally had to reuse one of these membrane. It worked.
But I never tried with a dried one.
thank you all, i'll try again, maybe this time i'll do primary antibody staining o/n at 4C (diluted in 5% BSA-
PBS-T).
thanks!
